LncRNA NEAT1 promotes Alzheimer's disease by down regulating micro-27a-3p

Abstract

Objective: Alzheimer's disease (AD) is a common neurodegenerative disease. This study was designed to investigate the roles of lncRNA NEAT1/miR-27a-3p axis in AD.

Methods: Amyloid protein was used to treat SH-SY5Y cells and rats to construct AD model. RT-qPCR was used to quantify lncRNA NEAT1 and micro-27a-3p in AD model cells. Western blot was used to determine the β-amyloid-precursor-protein-cleaver-enzyme 1 (BACE1), amyloid, Tau protein and its phosphorylation, Caspase 3 protein and its lytic cell protein and amyloid precursor protein (APP). Flow cytometry was used to detect apoptosis. The cell activity was detected by CCK-8. The lncRNA NEAT1 and miR-27a-3p inhibition or over-expression vectors were constructed. The dual luciferase reporter gene and RNA pull-down assay were used to detect the targeting relationship between lncRNA NEAT1 and micro-27a-3p. The cognitive function of rats was tested by water maze.

Results: After being induced by amyloid protein, lncRNA NEAT1 was up-regulated while micro-27a-3p was down-regulated in SH-SY5Y cells. Apoptosis rate was increased and cell activity was decreased. Amyloid protein, BACE1 protein, APP protein, Tau protein and its phosphorylation, Caspase 3 protein and its lytic cell protein were up-regulated. Down-regulation of lncRNA NEAT1 or up-regulation of micro-27a-3p could reduce cell apoptosis, increase cell activity, down-regulate amyloid protein, BACE1 protein, APP protein, Tau protein and its phosphorylation, and up-regulate caspase 3 protein and its lysate protein. Dual luciferase reporter gene assay and RNA pull-down experiments revealed that micro-27a-3p was the target gene of lncRNA NEAT1. Down-regulation of micro-27a-3p could offset the changes caused by LncRNA NEAT1. AD caused cognitive dysfunction in rats, which was improved by down-regulation of lncRNA NEAT1.

Conclusion: lncRNA NEAT1 regulates the development of AD by down-regulating micro-27a-3p.

Keywords: Alzheimer’s disease; lncRNA NEAT1; miR-27a-3p.

Figure 1

Amyloid was used to induce AD model in SH-SY5Y cells. A: Increased apoptosis was observed in SH-SY5Y cells after induction; B: Caspase 3 cleavage was observed in SH-SY5Y cells after induction; C: Western blot map; D: Up-regulation of AD related proteins BACE1, APP, Aβ, Tau and p-Tau was observed in SH-SY5Y cells after induction; E: Reduced cell proliferation was observed in SH-SY5Y cells after induction; F, G: Increased NEAT1 and reduced miR-27a-3p was observed in SH-SY5Y cells after induction. p-Tau, Tau protein phosphorylation. AD: Alzheimer’s disease. *P<0.05, **P<0.01, ***P<0.001.

Figure 2

Regulatory effect of NEAT1 on Ab-induced SH-SY5Y cells. A: NEAT1 siRNA down-regulated lncRNA NEAT1; B: Down-regulation of lncRNA NEAT1 led to the increase of cell activity; C: Down-regulating lncRNA NEAT1 reduced the apoptosis; D: Down-regulating lncRNA NEAT1 resulted in the down-regulation of Ab-related proteins BACE1, APP, Aβ, Tau and p-Tau; E: Down-regulation of lncRNA NEAT1 inhibited Caspase cleavage; F: Western blot. All the cells in the above experiments were induced by Ab to construct AD model. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

Regulatory effect of micro-27a-3p on Ab-induced SH-SY5Y cells. A: micro-27a-3p mimics up-regulated expression of micro-27a-3p; B: Up-regulation of micro-27a-3p caused the increase of cell activity; C: Up-regulation of micro-27a-3p led to the reduction of apoptosis; D: Up-regulation of micro-27a-3p caused the down-regulation of Ab-related proteins BACE1, APP, Aβ, Tau and p-Tau; E: Up-regulation of micro-27a-3p inhibited Caspase cleavage; F: Western blot. All the cells in the above experiments were induced by Ab to Alzheimer’s disease model. *P<0.05, **P<0.01, ***P<0.001.

Figure 4

micro-27a-3p was a downstream target of lncRNA NEAT1. A: Starbase 3.0 was used to predict the binding site between lncRNA NEAT1 and micro-27a-3p; B: Down-regulation of NEAT1 increased micro-27a-3p expression; C: Dual luciferase report; D: RNA pull-down experiment. CAP: m7GPPPN structure. UTR: untranslated area. AAA: ploy A tail. **P<0.01, ***P<0.001.

Figure 5

Offsetting effect between micro-27a-3p and lncRNA NEAT1. A: Down-regulation of micro-27a-3p offset the influence of lncRNA NEAT1 on apoptosis; B: Western blot; C: Down-regulation of micro-27a-3p offset the influence of lncRNA NEAT1 on AD-related proteins; D: Down-regulation of micro-27a-3p offset the influence of lncRNA NEAT1 on Caspase 3 cleavage; E: Down-regulation of micro-27a-3p offset the influence of lncRNA NEAT1 on cell activity.

Figure 6

lncRNA NEAT1 regulated cognitive function of AD rats in vivo through miR-27a-3p. A: Swimming speed of rats in each group. B: The number of crossing the platform of rats in each group. C: The time to reach the platform of rats in each group. D: Swimming path distance of rats in each group. E, F: Expression of NEAT1 and miR-27a-3p in hippocampus of rats in each group. Each group consisted of 6 rats (n=6).